Application of dihydroartemisinin in preparation of hemophilia arthropathy antioxidant medicine

Abstract

The invention discloses an application of dihydroartemisinin in preparation of an antioxidant drug for hemophilia arthropathy, and particularly relates to an application of DHA (docosahexaenoic acid) in delaying cartilage cell degeneration by inhibiting ROS (reactive oxygen species) level in cartilage cells in the presence of blood. In-experience verification shows that the DHA delays HA degeneration by delaying the ROS level in cartilage cells. Therefore, DHA has a good application prospect in treatment of hemophilia arthropathy. The extensive use of current modern hematology nursing does not provide considerable protection for the development of prevention of HA. The total knee arthroplasty can relieve pain of an end-stage HA patient and restore joint function. According to the invention, the oxidation resistance of the cartilage cells can be well improved, so that the progress of HA (hemagglutinin) is delayed.

Description

Technical Field

[0001] The invention relates to the technical field of dihydroartemisinin, and in particular to application of dihydroartemisinin in preparing an antioxidant drug for hemophilic arthropathy. Background Art

[0002] Hemophilic arthropathy (HA) is a debilitating disease caused by spontaneous intra-articular bleeding and recurrent intra-articular hematomas, with chronic pain, joint damage, and decreased quality of life as the main clinical manifestations. In the late stage, it is characterized by fibrosis and loss of function of the joints. The affected joints are mostly knees, elbows, and ankles. The typical pathological manifestations of HA are synovitis, articular cartilage destruction, and subchondral bone destruction. Since there is no treatment available to intervene in the continuation of HA, HA remains a persistent problem and challenge for hemophilia patients. Therefore, it is urgent to identify new therapeutic targets to slow down or even prevent cartilage degeneration.

[0003] A unique pathophysiological manifestation of HA is cartilage degeneration, which is an early target of HA development. Cartilage is a rather inert tissue composed of matrix proteins and chondrocytes. Since cartilage lacks a blood supply, it relies on synovial fluid for nutrition. The vulnerability of cartilage to blood is obvious. Even a single hemorrhage, which only exposes the superficial cartilage to blood, can lead to long-term changes in cartilage matrix turnover and ultimately its destruction. Because degenerative changes may not be observed clinically or radiographically until decades later, the underlying pathogenesis of articular cartilage damage is believed to be caused by blood. Studies have shown that monocytes / macrophages and red blood cells present in the blood can have direct deleterious effects on cartilage. In vitro and in vivo animal models have shown that short-term exposure of cartilage tissue to low concentrations of blood can lead to increased activity of matrix metalloproteinases (MMPs) and inhibition of proteoglycan synthesis. However, the molecular mechanisms by which joint hemorrhage leads to permanent impairment of chondrocyte activity and cartilage matrix turnover are not fully understood.

[0004] Excessive accumulation of reactive oxygen species (ROS) is a hallmark of chondrocytes after joint bleeds, leading to a persistent disturbance in cartilage matrix turnover. Iron from hemoglobin can catalyze the Fenton reaction to produce toxic hydroxyl radicals, leading to excessive production of ROS and oxidative stress in chondrocytes. After joint bleeds, blood monocytes in the joint cavity can produce interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) independently of the inflammatory response. These cytokines are known to have adverse effects on the turnover of cartilage matrix and promote the production of hydrogen peroxide by chondrocytes. Oxidative stress is the result of excessive production of ROS, which exceeds the ability of the cellular antioxidant defense system to effectively clear the cells. Excessive production of ROS in chondrocytes and the induction of oxidative stress are one of the main causes of osteoarthropathy. Many studies have shown that ROS levels are highly upregulated in human osteoarthropathy cartilage and chondrocytes. At the same time, stimulation of primary human chondrocytes and mouse chondrocytes with IL-1β increases the production of cellular and mitochondrial ROS, thereby promoting chondrocyte apoptosis, which is similar to what is observed in vivo. It has been reported that exposure of chondrocytes to H2O2, menaquinone, 3-morpholinosydnon imine (SIN1), tert-butyl peroxide (THBHP), or other prooxidants increases inflammation and apoptosis, suggesting that oxidative stress can induce chondrocyte inflammation. Increased oxidative stress is positively correlated with collagen degradation, suggesting the role of ROS in cartilage matrix catabolism. In addition, in different studies, it has been reported that NO and H2O2 inhibit the synthesis of proteoglycans, indicating the role of ROS in inhibiting cartilage matrix anabolism. In summary, oxidative stress has become an important part of HA research, and studies on the regulation of oxidative stress-related genes / pathways can provide new targets for the treatment of HA.

[0005] HA is a degenerative change of joints caused by repeated bleeding in the joints. Traditional Chinese medicine classifies HA as a "blood syndrome" or "blood disease". Its onset is caused by deficiency of Yin in the body, fumigation of fire and heat, forced blood flow, or failure of Qi to control blood, blood stasis blocking the collaterals and causing blood to overflow outside the vessels. Traditional Chinese medicine treats HA mainly by nourishing Yin, removing heat, cooling blood, stopping bleeding and removing blood stasis. Therefore, new understanding and breakthroughs are needed in the pathogenesis of HA, and new opportunities and ideas are also needed to explore the mechanism of intervention of the traditional Chinese medicine dihydroartemisinin (DHA) in HA. Summary of the invention

[0006] The purpose of the present invention is to provide an application of dihydroartemisinin in the preparation of an antioxidant drug for hemophilic arthropathy. Through the new function of dihydroartemisinin, that is, the function of having chondrocyte antioxidant activity, hemophilic arthropathy is effectively prevented and treated by increasing the antioxidant capacity of chondrocytes, thereby providing an application of dihydroartemisinin in the preparation of an antioxidant drug for hemophilic arthropathy.

[0007] The technical solution adopted by the present invention is:

[0008] The present invention provides an application of dihydroartemisinin in preparing an antioxidant drug for hemophilic arthritis; the drug is a chondrocyte antioxidant drug prepared by using dihydroartemisinin DHA; the dihydroartemisinin DHA can inhibit reactive oxygen species (ROS), and is used for preventing articular cartilage degeneration and delaying HA degeneration in hemophilic arthritis.

[0009] Specifically, in the presence of blood, dihydroartemisinin DHA delays chondrocyte degeneration by inhibiting the level of ROS in chondrocytes; it was verified that DHA delays the degeneration of HA by delaying the level of ROS in chondrocytes.

[0010] The active ingredient of the chondrocyte antioxidant drug is dihydroartemisinin.

[0011] The chemical formula of dihydroartemisinin DHA is C 15 H 24 O5, the structural formula is as follows:

[0012]

[0013] Specifically, the drug is administered orally.

[0014] Specifically, the drug is a drug that reduces the level of reactive oxygen species (ROS) in chondrocytes of F8 factor-deficient model mice and delays chondrocyte degeneration.

[0015] Specifically, the effective dose of dihydroartemisinin in delaying chondrocyte degeneration is 10 mg / kg.

[0016] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: 1) In the presence of blood, DHA delays the degeneration of chondrocytes by inhibiting the level of ROS in chondrocytes. 2) It is verified that DHA delays the degeneration of HA (hemophilic arthritis) by delaying the level of ROS in chondrocytes. Therefore, DHA has a good application prospect in the treatment of hemophilic arthritis. Due to the widespread adoption of modern hematological care, it does not provide considerable protection for the prevention of the development of HA. Even total knee replacement surgery to relieve pain and restore joint function in patients with end-stage HA. The present invention can well play its role in improving the antioxidant capacity of chondrocytes, thereby delaying the progression of HA. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0018] Figure 1This is a graph showing the effects of DHA intervention on the knee joints of mice in each group for 4 weeks; Figure 1 (A) is the Micro-CT scan result (Knee represents knee joint, subchondral bone, SB represents subchondral bone). Figure 1 (B) is the relative volume of bone tissue, Figure 1 (C) is the result diagram of trabecular bone thickness. Figure 1 (D) is the result diagram of trabecular separation;

[0019] Figure 2 The figure is the SO / FG staining and bone morphometric analysis of the knee joints of mice in each group after continuous DHA intervention for 4 weeks; Figure 2 (A) is the result of SO / FG staining scan. Figure 2 (B) is the OARSI score diagram of each knee joint;

[0020] Figure 3 The immunohistochemical staining results of the knee joints of mice in each group after continuous DHA intervention for 4 weeks are shown in the figure; Figure 3 (A) is Col2a1, Figure 3 (B) is the result of relative quantitative analysis of Col2a1. Figure 3 (C) is Mmp13, Figure 3 (D) is a graph showing the results of relative quantitative analysis of Mmp13;

[0021] Figure 4 The ROS staining results (A) and relative quantitative analysis (B) of the knee joints of mice in each group after continuous DHA intervention for 4 weeks;

[0022] Figure 5 The immunohistochemical staining results of the knee joints of mice in each group after continuous DHA intervention for 4 weeks. A is Nox1, B is the relative quantitative analysis result of Nox1, C is Nox2, and D is the relative quantitative analysis result of Nox2. E is Gpx4, and F is the relative quantitative analysis result of Gpx4;

[0023] Figure 6 This is a graph showing the effects of different concentrations of DHA on chondrocyte survival in the present invention;

[0024] Figure 7 The figure is a Real-time PCR analysis result of the mRNA expression of cartilage degeneration related genes under the blood induction state of DHA of the present invention; wherein AF are Adamts5, Mmp3, Mmp13, Col2a1, Aggrecan and Sox9 respectively;

[0025] Figure 8The immunofluorescence staining of cartilage degeneration related genes by DHA of the present invention under blood induction state; A is Col2a1, B is Mmp13, C is the relative quantitative analysis result of Col2a1, and D is the relative quantitative analysis result of Mmp13;

[0026] Fig. 9 The figure shows the effect of DHA on the protein expression of cartilage degeneration related genes under blood induction state; A is the Western blot diagram of chondrocytes in each group, B is the relative quantitative analysis result of Adamts5, C is the relative quantitative analysis result of Mmp3, D is the relative quantitative analysis result of Mmp13, E is the relative quantitative analysis result of Col2a1, F is the relative quantitative analysis result of Aggrecan, and G is the relative quantitative analysis result of Sox9;

[0027] Fig.10 The results of the DHA of the present invention on ROS activity in chondrocytes under blood induction (A) and relative quantitative analysis (B);

[0028] Fig.11 The figure is the effect of DHA of the present invention on SOD activity (A) and GSH level (B) in chondrocytes under blood induction state;

[0029] Fig.12 The figures are the results of the effect of DHA on the protein expression of oxidative stress-related genes under blood induction state of the present invention; A is the Western blot diagram of chondrocytes in each group, B is the relative quantitative analysis result diagram of Nox1, C is the relative quantitative analysis result diagram of Nox2, and D is the relative quantitative analysis diagram of Gpx4. DETAILED DESCRIPTION

[0030] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0031] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0032] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] Dihydroartemisinin used in the embodiments of the present invention: DHA (CAS: 71939-50-9, Chengdu Munster Biological Co., Ltd., China); PBS buffer pH 7.4 (Phosphate buffered saline, PBS, 10×, #46-013-CM, Mediatech, USA), the ultrapure water refers to water with a resistivity of 18 MΩ*cm (25°C), and the percentage concentrations described in the embodiments of the present invention are all mass percentage concentrations unless otherwise specified.

[0034] Example 1 In vivo study of dihydroartemisinin delaying HA progression by inhibiting oxidative stress levels

[0035] Objective To clarify the changes in ROS levels in chondrocytes regulated by DHA under pathological conditions and to clarify the role of DHA in preventing and treating HA.

[0036] 1. Methods

[0037] 1 Experimental Grouping

[0038] (A) Control group; (B) Model group; (C) Low-dose drug intervention group (DHA-L group); (D) High-dose drug intervention group (DHA-H group). 10 mice in each group.

[0039] 2 Establishment of mouse model of intraarticular bleeding

[0040] The method for constructing the F8 factor-deficient joint cavity bleeding model mouse is as follows: 10-week-old male adult F8 factor-deficient transgenic mice were selected, and the F8 factor-deficient mice were anesthetized with 3% isoflurane and maintained with 1% isoflurane. Joint bleeding was simulated by acupuncture in the medial side of the right knee patellar ligament toward the joint capsule with a 30-gram insulin needle. The left knee joint of the F8 factor-deficient mice in the model group was used as the control group.

[0041] 3. Drug intervention

[0042] Three days after modeling, the mice in the DHA group were given the Chinese medicine monomer component DHA (10 mg / Kg or 20 mg / Kg, prepared in corn oil) by daily gavage for 4 consecutive weeks, and the mice in the control group were given the same dose of corn oil by gavage.

[0043] 4 indicators detection

[0044] 4.1 Micro-CT detection

[0045] The knee joint tissue of mice was taken 4 weeks after modeling, the surrounding muscle tissue was stripped, and fixed with 4% polyformaldehyde for 3 days. The fixed tissue specimens were placed on the μCT mouse bed along the sagittal direction for x-ray scanning. μCT scanning (Skyscan1272, Bruker, USA), the scanning software was SkyScan1176, the resolution was 8.73μm, the voltage was 42kV, the current was 555μA, the filter was Al 0.2mm, and the spiral scanning was 180°. The onboard CTvox graphics software was used for three-dimensional image construction. Finally, the onboard bone micromorphology analysis software CTAn was used to quantitatively analyze the region of interest (ROI). The 3D morphometric parameters include: relative trabecular volume (Bone volume / total volume, BV / TV), trabecular thickness (Trabecular Thickness, Tb.Th) and trabecular separation (Trabecular Separation, Tb.Sp).

[0046] 4.2 Histological and bone morphometric analysis

[0047] 4.2.1 Obtaining knee tissue and preparing slices from F8-deficient mice

[0048] After the Micro-CT scan, the knee joint samples of each group of mice were taken out, rinsed with tap water for 30 minutes, and then decalcified with 14% EDTA solution for 14 days, with the solution changed every day. After decalcification, they were rinsed with tap water for two hours, and then processed by a fully automatic tissue dehydrator and embedded in paraffin to prepare wax blocks. When slicing, the slices were sliced ​​at a thickness of 3 μm, and 15 slices were cut for each sample.

[0049] 4.2.2 Safranin / Fast Green (SO / FG) staining

[0050] 1) Take slices of knee joint tissue from each group (slices No. 1, 5, 10, and 15 of each sample) and bake at 60°C overnight;

[0051] 2) The next day, remove the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (dewaxing with xylene 3 times, 5 minutes each time; 100% and 95% alcohol 2 times, 5 minutes each time; 70% alcohol 1 time, 5 minutes each time; deionized water 2 times, 1 minute each time);

[0052] 3) Wash the sections in PBS three times, 3 minutes each time;

[0053] 4) After staining the sections in 0.5% Fast Green for 5 minutes, rinse with deionized water several times until there is no green in the cartilage (approximately 3 times);

[0054] 5) Place the slices in 0.5% acetic acid for 10 seconds, then wash with deionized water three times, 3 minutes each time;

[0055] 6) Place the sections in 2.5% SO stain for 2 minutes and wash with deionized water 5 times, 3 minutes each time;

[0056] 7) Place the slices in 95% alcohol for 3 seconds;

[0057] 8) Dehydrate and make transparent step by step (95% alcohol 3 times, 1 minute / time; 100% alcohol 2 times, 1 minute / time; xylene dewaxing 3 times, 1 minute / time), and seal with sealing agent. Observe and complete the slide under Zeiss upright microscope, the results are shown in Figure 2 Middle A.

[0058] 4.2.3 Bone morphometry

[0059] The morphological changes of the knee joint cartilage in each group were observed and compared, and bone tissue morphometry analysis was performed. The slices of the knee joint tissue in each group (slices 1, 5, 10, and 15 of each sample) were taken, and the cartilage area of ​​each knee joint sample was measured using Image J 1.46r software. The results are shown in Figure 2 Middle (B).

[0060] 4.2.4 OARSI scoring according to OA severity grading

[0061] Slice images of knee joint tissues from each group (slices 1, 5, 10, and 15 of each sample) were taken to evaluate the severity of OA cartilage damage using the histological scoring system developed by the Osteoarthritis Research Society International (OARSI) (OARSI scores are shown in Table 1). Two people other than the project team members graded the severity of OA cartilage damage and took the average of the scores. Figure 2 Middle (C).

[0062] 4.3 Immunohistochemical staining

[0063] Tissue sections of the knee joints were taken from each group and baked at 60°C overnight. The next day, the sections were taken out and dewaxed and rehydrated step by step after returning to room temperature (xylene dewaxing 3 times, 5 minutes / time; 100% and 95% alcohol 2 times, 5 minutes / time; 70% alcohol 1 time, 5 minutes / time; deionized water 2 times, 1 minute / time). All antibodies were heat-fixed as follows: heat-fixed with sodium citrate (pH 6.0) (temperature 60°C), and the sections were washed 3 times with 1× PBS for 3 minutes / time after returning to room temperature. After repair, incubate with 50-100u endogenous peroxidase blocker for 15 minutes, wash 3 times with 1×PBS, 3 minutes each time; add Col2a1 (1:200), Mmp13 (1:200), Nox1 (1:200), Nox2 (1:200), Gpx4 (1:200) primary antibodies (diluted with PBS containing 5% NGS and 0.1% Triton X-100 detergent), and then place the sections in a humidified box and incubate overnight at 4°C. The next day, the wet box was removed and the sections were washed 3 times with 1×PBS after returning to room temperature naturally, for 5 minutes each time. The corresponding secondary antibody (1×PBS diluted 1:1000) was added, and the sections were incubated at room temperature for 1 hour, and then washed 3 times with 1×PBS, for 5 minutes each time. Nuclear counterstaining was performed with hematoxylin for 10 seconds, and the sections were washed 3 times with deionized water, for 5 minutes each time. The sections were dehydrated and transparent step by step (95% alcohol 3 times, 1 minute each time; 100% alcohol 2 times, 1 minute each time; xylene dewaxing 3 times, 1 minute each time), and the sections were sealed with sealing agents. The sections were observed and collected under a Zeiss upright microscope, and the comparison analysis was performed. The results are shown in the figure. Figure 3 and Figure 5 .

[0064] 4.4 Organizing ROS detection

[0065] Take the tissue sections of the knee joint from each group and bake the sections at 60°C overnight. The next day, take out the sections, wait for them to return to room temperature, and then dewax and rehydrate them step by step (xylene dewaxing 3 times, 5 minutes / time; 100% and 95% alcohol 2 times, 5 minutes / time; 70% alcohol 1 time, 5 minutes / time; deionized water 2 times, 1 minute / time). Add 200μl of cleaning solution working solution, cover the entire surface of the section, and let it stand for 3-5 minutes. Aspirate the cleaning solution. Then add 100-200μl of staining probe working solution (1:500 dilution) and incubate in a 37°C incubator away from light for 30 minutes. Remove the staining solution and wash the sections with PBS 2-3 times. Use the sealing agent to complete the sealing. Observe and complete the slice collection under a Zeiss upright microscope, and perform control analysis. The results are shown in the table below. Figure 4 .

[0066] 2. Results

[0067] 1Micro-CT test results

[0068] The results of 3D reconstruction of Micro-CT showed that 4 weeks after modeling, the model group had obvious osteophytes around the knee joint and severe subchondral bone loss, while the DHA group had significantly less osteophytes around the knee joint. In addition, the subchondral osteoporosis was relatively mild (e.g. Figure 1 3D morphometric analysis showed that the BV / TV and Tb.Th of the medial subchondral bone of the knee joint in the DHA group were lower than those in the model group, while Tb.Sp was higher than that in the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05). Figure 1 In (such as Figure 1 (B) Figure 1 (C) and Figure 1 (as shown in (D)).

[0069] 2 Histological and bone morphometric analysis results

[0070] SO / FG staining results showed that the articular cartilage of the mice in the model group was destroyed and the cartilage layer was thinned 4 weeks after modeling, while the cartilage of the mice in the DHA group was partially destroyed after 4 weeks of DHA intervention, but relatively intact, and the cartilage layer was significantly increased compared with the model group (e.g. Fig.12 (A)). In addition, bone tissue morphometric analysis showed that after 4 weeks of DHA intervention, the knee cartilage area of ​​the DHA group was significantly increased compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better treatment effect than the DHA-H group, and the difference was statistically significant (p<0.05) (as shown in Fig.12 (B)). The results of the OARSI scoring system showed that the OARSI score of the knee cartilage of the DHA group mice was significantly lower than that of the model group after 4 weeks of DHA intervention; the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) ( Figure 2 (A) Figure 2 (B) Figure 2 (as shown in (C)).

[0071] 3 Immunohistochemical staining and immunofluorescence detection results

[0072] The results of Col2a1 immunohistochemical staining showed that the positive expression of Col2a1 in the model group decreased significantly 4 weeks after modeling, while the positive expression of Col2a1 in the DHA group increased significantly after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05). Figure 3 (A) Figure 3(C)); The results of Mmp13 immunohistochemical staining showed that the positive expression of Mmp13 in the model group increased significantly 4 weeks after modeling, while the positive expression of Mmp13 in the DHA group was significantly reduced after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) (as shown in Figure 3 (B) Figure 3 (D)). In addition, ROS immunofluorescence detection found that the ROS level in chondrocytes of the model group increased significantly 4 weeks after modeling, while the ROS level in chondrocytes of the DHA group mice decreased significantly after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) ( Figure 4 (A) Figure 4 (B)). Nox1 immunohistochemical staining results showed that the positive expression of Nox1 in the model group increased significantly 4 weeks after modeling, while the positive expression of Nox1 in the DHA group was significantly reduced after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) ( Figure 5 (A) Figure 5 (D)); Nox2 immunohistochemical staining results showed that Nox2 positive expression in the model group increased significantly 4 weeks after modeling, while Nox2 positive expression in the DHA group was significantly reduced after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) (as shown in Figure 5 (B) Figure 5 (E)); Gpx4 immunohistochemical staining results showed that the positive expression of Gpx4 in the model group was significantly reduced 4 weeks after modeling, while the positive expression of Gpx4 in the DHA group was significantly increased after 4 weeks of DHA intervention compared with the model group, and the difference was statistically significant (p<0.05); the DHA-L group had a better therapeutic effect than the DHA-H group, and the difference was statistically significant (p<0.05) (as shown in Figure 5 (C) Figure 5 (F)).

[0073] Conclusion

[0074] 1DHA can effectively improve the degeneration of cartilage and subchondral bone and delay the progression of HA.

[0075] 2DHA delays chondrocyte degeneration by inhibiting the level of oxidative stress in chondrocytes, thereby delaying the degeneration of HA.

[0076] Table 1 OARSI scoring details

[0077]

[0078] Example 2 In vitro study on the effect of dihydroartemisinin on preventing chondrocyte degeneration by regulating oxidative stress levels

[0079] Objective To verify in vitro that DHA regulates the changes in oxidative stress levels in chondrocytes under blood-induced conditions and to elucidate the mechanism by which DHA prevents chondrocyte degeneration.

[0080] 1. Methods

[0081] 1 Experimental Grouping

[0082] (A) Control group; (B) Blood group; (C) Blood+1μM DHA group; (D) Blood+5μM DHA group; (E) Blood+10μM DHA group.

[0083] 2 Isolation and culture of mouse articular chondrocytes

[0084] 1) A 10-day-old C57BL / 6J mouse was killed with CO2 and then immersed in 70% alcohol for 5-10 minutes.

[0085] 2) The mouse was removed, the skin was removed, the femoral head cartilage layer was grasped with forceps, and the attached ligaments and soft tissues were cut with scissors. The cartilage tissue was placed in a 1.5 mL centrifuge tube containing PBS.

[0086] 3) Remove the absorbent pad, clean the clean bench, change gloves, and preheat the digestion medium (F12 medium + 10% FBS + 1% P / S + collagenase P 0.5 mg / mL) at 37°C.

[0087] 4) Wash the cartilage cap in the 1.5 mL centrifuge tube with PBS 4 times, pipetting more than 10 times each time.

[0088] 5) Discard the PBS supernatant, add digestion medium, and flush out the cartilage tissue into a new 10 cm culture dish. Repeat the flushing several times to ensure that the digestion medium completely covers the cartilage tissue.

[0089] 6) Gently shake the culture dish, mark it, and place it in a 37℃ incubator for digestion for 4-6 hours.

[0090] 7) The cartilage tissue was blown every hour. After the end, the digestion medium in the culture dish was transferred to a 15 mL centrifuge tube and continued to be blown (30-40 times). After filtering with a 40 μm cell strainer, centrifuge at 1000 rpm for 5 minutes.

[0091] 8) Resuspend with 5 mL of normal culture medium (F12 medium + 10% FBS + 1% P / S), count and adjust the appropriate concentration to inoculate in a well plate, place in a 37°C, 5% CO2 constant temperature incubator for 12 hours, and continue with subsequent experiments after the cells are well attached to the wall.

[0092] 3. Blood extraction

[0093] Blood collected by cardiac puncture from 10-week-old C57BL / 6J mice was placed in 7.5 mM EDTA and centrifuged to separate plasma and cells (10 000 g, 10 min, Eppendorf). EDTA-treated cells were frozen at -80°C and thawed before adding chondrocytes (blood cells 2×107 μL / mL) for 24 h.

[0094] Effect of 4DHA on the survival of primary articular chondrocytes in mice

[0095] After the cells adhered, the blank group (no cells and no DHA) and the control group (cells added but no DHA) were replaced with serum-free culture medium (F12 culture medium), and the drug group was added with serum-free culture medium with the following final concentrations of DHA (1μM, 5μM, 10μM, 20μM, 50μM, 100μM), and culture was continued for 24 hours and 48 hours, respectively. At the corresponding time, the culture medium was aspirated, and the cells were gently washed twice with DPBS. 100μL of F12 culture medium containing 10μL CCK8 was added to each well. After continuing to culture for 2 hours, the supernatant was collected, and the average optical density (OD value) of each well was measured at 450nm. The results were repeated 3 times independently. Calculate the survival rate of chondrocytes. Chondrocyte survival rate (%) = [(OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. See the results. Figure 6 (A) and Figure 6 (B).

[0096] 5Real-time PCR analysis of mRNA expression levels of cartilage degeneration-related genes in chondrocytes of each group

[0097] 5.1 Extraction of mRNA from primary chondrocytes

[0098] 1) Aspirate the original culture medium, wash once with 1× PBS, add 0.5 mL Trizol directly to the culture plate, pipette and vortex to mix evenly, transfer to a separation gel tube (centrifuge at 12,000 rpm for 1 minute before use), and let stand at room temperature for 5 minutes.

[0099] 2) Add 0.1 mL of chloroform (1 / 5 of the total volume of Trizol) to the gel tube in step 1), mix by inverting for 15-30 seconds, and let stand at room temperature for 2-3 minutes; after completion, centrifuge at 12,000 rpm at 4°C for 15 minutes, carefully aspirate the supernatant, and transfer it to another 1.5 mL centrifuge tube.

[0100] 3) Add 1 volume of 70% alcohol to the supernatant of step 2), shake and mix for 30 seconds, add it to the 2 mL collection tube provided by the RNA extraction kit, and further purify the mRNA according to the operating instructions in the kit.

[0101] 5.2 mRNA concentration determination and reverse transcription

[0102] 1.5 μL of mRNA was taken from each group of samples, and the RNA concentration was measured using an ultra-micro UV spectrophotometer; 500 ng of mRNA was taken from each group of samples to prepare the reaction system (10 μL) according to the operating instructions of the reverse transcription kit to synthesize cDNA, and the remaining mRNA was stored in a -80°C refrigerator.

[0103] The reverse transcription reaction system was as follows: 500 ng mRNA, 2 μL 5x Mix, and RNase-free water to a volume of 10 μL.

[0104] The reverse transcription procedure was as follows: reverse transcription reaction at 37°C for 15 minutes, reverse transcriptase inactivation (termination reaction) at 85°C for 5 seconds.

[0105] 5.3 Real-time PCR quantitative analysis of genes

[0106] Take the complementary deoxyribonucleic acid (cDNA) obtained by reverse transcription in each group, add 40 μL of RNase-free water and dilute it 5 times as a PCR reaction template. Take 500 ng of mRNA from each group of samples and prepare the reaction system (10 μL) according to the operating instructions in the SYBR kit to amplify the target gene, and the remaining mRNA is stored in a -80°C refrigerator. In this experiment, Adamts5, Mmp3, Mmp13, Col2a1, Aggrecan, and Sox9 were selected as target genes, and Actb was used as the internal reference gene (primer sequences are shown in Table 2). The target gene copy number / Actb ratio was used as the statistical value, and the 2-ΔΔCT method was used to analyze the real-time fluorescence PCR data. Real-time PCR was used to analyze the expression level of the target gene in each group of primary chondrocytes. The results are shown in Figure 7 shown.

[0107] The real-time PCR reaction system is as follows:

[0108] 2 μL of cDNA template, 0.5 μL of upstream primer (10 μM), 0.5 μL of downstream primer (10 μM), 5 μL of 2x Sybr green mix, and RNase-free water to make up to 10 μL.

[0109] Real-time PCR reaction procedure is as follows:

[0110] Initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 60 seconds (40 cycles of denaturation, annealing and extension), and melting curve at 60-95°C for 5 seconds hold / 1°C.

[0111] Table 2 Primer sequences

[0112]

[0113]

[0114] 6. Cell immunofluorescence staining detection

[0115] For immunofluorescence staining, the cells were fixed with 4% cell fixative at 37°C for 30 minutes, washed 3 times with PBS for 3 minutes each time, permeabilized with 0.03% Triton-X for 10 minutes, and then incubated with 5% goat serum for 1 hour, incubated with primary antibodies of Col2a1 (1:1000) and Mmp13 (1:100) at 4°C overnight, and then incubated with Alexa FluorTM 488-conjugated goat anti-rabbit antibody (1:1000) for 1 hour and stained with DAPI (1:100) for 10 minutes. Finally, positive cells were detected by fluorescence microscopy. Quantitative analysis was performed by ImageJ software. Results are shown in Figure 8 .

[0116] 7. Western-Blot detection of the expression of cartilage degeneration-related proteins in human chondrocytes under blood conditions

[0117] 7.1 Total protein extraction from articular chondrocytes

[0118] 1) Aspirate the culture medium of each group in the 6-well plate, wash once with pre-cooled 1× PBS, add pre-cooled cell lysis buffer (containing 1% protease phosphatase inhibitor), let it stand for 5 minutes, carefully scrape the cells from the culture plate with a cell scraper, blow into a cell suspension and transfer to a 1.5 mL centrifuge tube, let it stand on ice for 30 minutes, and vortex every 10 minutes.

[0119] 2) Centrifuge at 4°C and 12,000 rpm for 10 minutes, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, and place on ice to obtain the total protein extract.

[0120] 7.2 Quantification of total protein in articular chondrocytes

[0121] 1) Add 1 mL of the protein sample to be tested, cell lysis buffer (containing 1% protease phosphatase inhibitors), and 1×PBS into labeled 1.5 mL centrifuge tubes and place them together in crushed ice for later use.

[0122] 2) Preparation of standard test solutions: 2 mg / mL BSA standard (10×1 mL ampules, containing 2 mg / mL bovine serum albumin (BSA), 0.9% salt and 0.05% sodium azide) was diluted with cell lysis buffer (containing 1% protease phosphatase inhibitors) to obtain protein standard test solutions with BSA concentrations of 2000 μg / mL, 1500 μg / mL, 1000 μg / mL, 750 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, and 0 μg / mL.

[0123] 3) Preparation of BCA working solution: According to the number of standard substances and samples to be tested, add BCA reagent A:BCA reagent B in the BCA protein concentration kit (#23225, Thermo Fisher Scientific, USA) into the sample adding tank at a ratio of 50:1 and mix well. The solution is stable at room temperature for 24 hours.

[0124] Total volume of BCA working solution = (number of standards + number of samples) × 2 replicate wells × 200uL

[0125] 4) Take 10uL of protein standard test solution and the sample test solution prepared in 3.5.1 and add them to the standard well and sample well of a 96-well ELISA plate respectively. Prepare 2 wells in parallel for each concentration.

[0126] 5) Add 200uL BCA working solution to each well, cover, and incubate at 37°C for 30 minutes.

[0127] 6) After the incubation, the cells were allowed to return to room temperature naturally and placed in a cell imaging microplate detection system. The OD value was measured at λ = 562 nm to prepare a standard curve. Figure 1 As shown, the regression equation is: y(OD)=0.0005x(C)+0.0059, R 2 =0.9983, that is, the concentration of the protein sample to be loaded = (OD-0.0059) / 0.0005.

[0128] 7.3 Polyacrylamide gel electrophoresis of total protein in articular chondrocytes

[0129] 1) Prepare equal volumes and masses of protein samples to be loaded according to the protein concentrations of each group measured above. Take 20ug protein from each group of samples, calculate its volume, fill it up to 16uL with cell lysis buffer (containing 1% protease phosphatase inhibitors), and finally add 4uL 5× loading buffer, with a final volume of 20uL. Add three-color pre-stained markers at the same time.

[0130] 2) Incubate each group of protein samples in a metal bath at 100°C for 10 minutes, return to room temperature, centrifuge briefly, and place on ice for later use.

[0131] 3) Take the precast protein gel, carefully pull out the "comb" of the gel plate, and tear off the green seal at the bottom; insert the gel plate into the vertical electrophoresis tank containing 1× electrophoresis buffer.

[0132] 4) Add 5uL of protein marker to well 1 of the gel, and add 20uL of the protein sample to be loaded to the remaining wells.

[0133] 5) After the sample is loaded, insert the electrode into the appropriate position of the vertical electrophoresis tank; turn on the power of the electrophoresis instrument, adjust the voltage to 80V, wait for 30 minutes, adjust the voltage to 100V, wait for 1 hour, until 10kDa reaches the bottom of the gel, and stop the electrophoresis instrument.

[0134] 7.4 Total protein transfer of articular chondrocytes

[0135] 1) Take out the gel sheet from the vertical electrophoresis tank, rinse the gel sheet with tap water, carefully remove the short gel sheet, cut off the excess gel with a gel knife, carefully transfer the gel from the long gel sheet to deionized water, and soak for later use.

[0136] 2) Take a small PVDF transfer package, place the transfer layer containing PVDF membrane on the electrode plate, and be careful to remove bubbles; carefully transfer the glue from deionized water to the PVDF membrane, and be careful to remove bubbles; then place the transfer layer without PVDF membrane on the PVDF membrane, and be careful to remove bubbles. After completion, cover the electrode cover, insert the transfer plate into the transfer instrument, and transfer for 1 hour at 0.4A and 80V.

[0137] 7.5 Western Blotting of Articular Chondrocytes

[0138] 1) Blocking: After the transfer is completed, the protein-loaded PVDF membrane is quickly placed in 3-5 mL of blocking solution (1×TBST containing 5% skim milk) and shaken at room temperature for 1 hour.

[0139] 2) Primary antibody incubation: After blocking, add appropriate amount of Adamts5, Mmp3, Mmp13, Col2a1, Aggrecan, Sox9 primary antibodies into the blocking solution according to the dilution factor (1:1000), mix well, and place on a shaker at 4°C overnight.

[0140] 3) Secondary antibody incubation: The next day, remove the primary antibody blocking solution, add an appropriate amount of 1×TBST and wash three times for 10 minutes each; add an appropriate amount of blocking solution, dilute it according to the specific dilution factor (1:5000), add an appropriate amount of corresponding secondary antibody to the blocking solution, mix well, and shake at room temperature for 1 hour.

[0141] 4) Development: After the secondary antibody incubation, remove the secondary antibody blocking solution, add an appropriate amount of 1×TBST and wash three times, 10 minutes each time; carefully absorb excess water with absorbent paper, quickly place it in a transparent paper protective cover, add premixed ECL developer solution, make it evenly distributed on the test membrane, and remove excess bubbles; slightly absorb excess ECL developer solution with absorbent paper; place the test membrane in the Image Quant LAS 4000 (EG, USA) imaging system for imaging, and use ImageJ for semi-quantitative analysis.

[0142] 5) Antibody elution: After development, transfer the membrane to be tested to an incubation box containing TBST, wash 3 times, 5 minutes each time; add 1× antibody elution buffer for 15 minutes; then add TBST for 3 times, 5 minutes each time.

[0143] 6) Blocking: After washing is completed, the membrane to be tested is quickly placed in 3-5 mL of blocking solution (1×TBST containing 5% BSA) and shaken for 1 hour.

[0144] 7) Incubation of internal reference protein β-actin: After blocking, add an appropriate amount of β-actin primary antibody to the blocking solution according to the dilution factor (1:2000), mix thoroughly, and shake on a shaker at 4°C overnight. The remaining steps are the same as 2)-4). The results are shown in Fig. 9 .

[0145] 8 Detection of ROS levels in primary chondrocytes

[0146] The cells were processed and the DCFH-DA probe was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 μmol / L. The cell culture medium was removed and an appropriate volume of the diluted DCFH-DA was added. The volume added should be sufficient to cover the cells. Incubate in a cell culture incubator at 37°C for 20 minutes. Then, positive cells were detected by fluorescence microscopy. Quantitative analysis was performed using ImageJ software. The results are shown in Fig.10 .

[0147] 9 Detection of SOD activity in primary chondrocytes

[0148] 9.1 Remove the culture medium from each group in the 6-well plate, wash once with pre-cooled 1× PBS, add pre-cooled PBS, let stand for 5 minutes, carefully scrape the cells from the culture plate with a cell scraper, blow into a cell suspension and transfer to a 1.5mL centrifuge tube, break the cells in an ultrasonic ice bath. Then, centrifuge at 12,000rpm and 4℃ for 15 minutes, carefully aspirate the supernatant and transfer it to another 1.5mL centrifuge tube.

[0149] 9.2 Preparation of BCA working solution: According to the number of samples, add BCA reagent A: BCA reagent B in the BCA protein concentration kit (#23225, ThermoFisher Scientific, USA) into the sample adding tank at a ratio of 50:1 and mix well. The solution is stable at room temperature for 24 hours.

[0150] Total volume of BCA working solution = (number of standards + number of samples) × 2 replicate wells × 200uL;

[0151] 9.3 Take 10uL of protein standard test solution and the sample test solution prepared in 3.5.1 and add them to the standard wells and sample wells of a 96-well ELISA plate respectively. Prepare 2 wells in parallel for each concentration.

[0152] 9.4 Add 200uL BCA working solution to each well, cover, and incubate at 37℃ for 30 minutes.

[0153] 9.5 After the incubation, allow it to return to room temperature naturally, place it in the cell imaging microplate detection system, measure the OD value at λ = 562nm, and make a standard curve as shown in Figure 1 As shown, the regression equation is: y(OD)=0.0005x(C)+0.0059, R 2 =0.9983, that is, the concentration of the protein sample to be loaded = (OD-0.0059) / 0.0005.

[0154] 9.6 Preparation of SOD standard: Use the SOD detection buffer provided in this kit to dilute the SOD standard to the following concentration series: 200U / ml, 100U / ml, 50U / ml, 20U / ml, 10U / ml, 5U / ml, 2U / ml.

[0155] 9.7 Preparation of SOD detection working solution and reaction starting solution: According to the number of standard substances and samples to be tested, mix them together according to the proportions in the SOD detection kit (S0109, Biotime, China).

[0156] 9.8 Total volume of SOD detection solution = (number of standards + number of samples) × 2 replicate wells × 160uL; Total volume of SOD reaction starting solution = (number of standards + number of samples) × 2 replicate wells × 20uL.

[0157] 9.9 Take 20uL of protein standard test solution and sample test solution prepared in 3.5.1 and add them to the standard wells and sample wells of 96-well ELISA plate respectively. Prepare 2 wells in parallel for each concentration.

[0158] 9.10 Add 160uL SOD detection solution, 20uL SOD reaction start solution and 20uL sample to each well, cover and incubate at 37℃ for 30 minutes.

[0159] 9.11 After the incubation, allow it to return to room temperature naturally, place it in the cell imaging microplate detection system, measure the OD value at λ = 600nm, and make a standard curve as shown below: Figure 1 As shown, the regression equation is: y(OD)=0.091x(C)-0.0856, R 2 =0.9938, that is, the concentration of the protein sample to be loaded = (OD+0.0856) / 0.091.

[0160] 9.12 Calculation of total SOD activity in the sample: Inhibition percentage = [(A blank control 1 - A blank control 2) - (A sample - A blank control 3)] / (A blank control 1 - A blank control 2) × 100%; SOD enzyme activity units in the sample to be tested = SOD enzyme activity units in the detection system = inhibition percentage / (1 - inhibition percentage) units.

[0161] 10 Detection of GSH levels in primary chondrocytes

[0162] 10.1 Aspirate the culture medium, wash the cells once with PBS, collect the cells by centrifugation, and aspirate the supernatant. Add 3 times the volume of the cell pellet to the protein removal reagent M solution, and fully vortex. Then use liquid nitrogen and a 37°C water bath to quickly freeze and thaw the sample twice. Place it at 4°C or in an ice bath for 5 minutes. Centrifuge at 10,000g for 10 minutes at 4°C. Take the supernatant for the determination of total glutathione.

[0163] 10.2 Preparation of Standards

[0164] 1) Dilute the 10mM GSSG stock solution with protein removal reagent M solution to make a 15μM GSSG solution. Then dilute it to 10, 5, 2, 1, and 0.5μM GSSG solutions in sequence. Take six points of 15, 10, 5, 2, 1, and 0.5μM GSSG solutions to make a standard curve.

[0165] 2) If the GSSG content in the sample needs to be determined, add the diluted GSH scavenging auxiliary solution at a ratio of 20 μl of diluted GSH scavenging auxiliary solution per 100 μl of standard, and vortex mix immediately. Then add the GSH scavenging reagent working solution at a ratio of 4 μl of GSH scavenging reagent working solution per 100 μl of standard, vortex mix immediately, and react at 25°C for 60 minutes. It can be used for subsequent GSSG content detection.

[0166] 10.3 Determination of samples and standards

[0167] 1) Use a 96-well plate, add samples or standards in sequence, mix well. Add 150 μl of total glutathione detection working solution, mix well, and incubate at 25°C or room temperature for 5 minutes.

[0168] 2) Add 50 μl of 0.5 mg / ml NADPH solution and mix well;

[0169] 3) Preparation of total glutathione detection working solution: According to the number of samples to be detected, refer to the table below to prepare an appropriate amount of total glutathione detection working solution. The total glutathione detection working solution is obtained by mixing the three reagents of protein removal reagent M, total glutathione detection solution and 0.5 mg / ml NADPH in proportion.

[0170] 4) Total volume of GSH detection solution = (number of standards + number of samples) × 2 replicate wells × 200uL.

[0171] Incubate at room temperature for 25 minutes and measure the OD value at A=412 nm.

[0172] 10.4 Calculation of total glutathione content in samples

[0173] The calculation formula is: GSH = Total Glutathione-GSSG × 2 (Note: Total Glutathione is the GSSG concentration calculated by the standard curve multiplied by 2. At the same time, the GSSG obtained after removing GSH should also be multiplied by 2, because 1 GSSG molecule can be reduced to 2 GSH molecules after the reaction). The results are shown in Fig.11 Middle B.

[0174] 11. Western-Blot detection of the expression of proteins related to oxidative stress in human chondrocytes under blood conditions

[0175] The extraction, quantification, electrophoresis, membrane transfer and protein immunoblotting of total protein in articular chondrocytes were the same as in Experimental Method 6 of Example 2. The expression levels of Nox1, Nox2 and Gpx4 proteins were detected respectively. The results are shown in Fig.12 .

[0176] 2. Results

[0177] Effect of 1DHA on the survival of mouse articular chondrocytes

[0178] In this study, the CKK8 method was used to determine the effect of different concentrations of DHA on chondrocyte survival. The survival rate of primary mouse articular chondrocytes under the action of different concentrations of DHA is shown in Figure 2. Figure 6 As shown in , the survival rate of mouse primary articular chondrocytes was not affected by different concentrations of DHA. Therefore, in subsequent experiments, we used 1μM, 5μM and 10μM DHA for subsequent studies.

[0179] mRNA and protein expression levels of chondrocyte degeneration-related genes after 2DHA intervention

[0180] Figure 7The results showed that compared with the control group, the mRNA levels of Adamts5, Mmp3 and Mmp13 in chondrocytes of the blood-induced group were significantly increased, while the mRNA levels of Col2a1, Aggrecan and Sox9 were significantly decreased. The differences were statistically significant (p<0.05). Compared with the blood-induced group, after DHA treatment, the mRNA levels of Adamts5, Mmp3 and Mmp13 decreased in a dose-dependent manner, while the mRNA levels of Col2a1, Aggrecan and Sox9 increased in a dose-dependent manner, and the differences were statistically significant (p<0.05). In addition, Figure 8 and Fig. 9 As shown in the figure, compared with the control group, the expression of Adamts5, Mmp3 and Mmp13 proteins in chondrocytes of the blood-induced group was significantly increased, while the expression of Col2a1, Aggrecan and Sox9 proteins was significantly decreased. The differences were statistically significant (p<0.05). Compared with the blood-induced group, after DHA treatment, the expression of Adamts5, Mmp3 and Mmp13 proteins decreased in a dose-dependent manner, while the expression of Col2a1, Aggrecan and Sox9 proteins increased in a dose-dependent manner, and the differences were statistically significant (p<0.05).

[0181] Effects of 3DHA intervention on oxidative stress-related indicators in chondrocytes

[0182] Fig.10 and Fig.11 As shown in the figure, compared with the control group, the ROS level in the chondrocytes of the blood-induced group increased significantly, while the SOD activity and GSH level decreased significantly. The differences were statistically significant (p<0.05). Compared with the blood-induced group, after DHA treatment, the ROS level decreased in a dose-dependent manner, while the SOD activity and GSH level increased in a dose-dependent manner, and the differences were statistically significant (p<0.05). Fig.12 As shown in the figure, compared with the control group, the expression of Nox1 and Nox2 proteins in chondrocytes of the blood-induced group was significantly increased, while the expression of Gpx4 protein was significantly decreased. The differences were statistically significant (p<0.05). Compared with the blood-induced group, after DHA treatment, the expression of Nox1 and Nox2 proteins decreased in a dose-dependent manner, while the expression of Gpx4 protein increased in a dose-dependent manner, and the differences were statistically significant (p<0.05).

[0183] Conclusion

[0184] Under the blood-induced state, DHA delayed chondrocyte degeneration by inhibiting the level of oxidative stress.

[0185] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application.

[0186] It should be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. A use of dihydroartemisinin in the preparation of an antioxidant drug for hemophilic arthropathy, characterized in that: The drug is a chondrocyte antioxidant drug prepared by using dihydroartemisinin DHA; the dihydroartemisinin DHA can inhibit reactive oxygen species (ROS), and is used to prevent articular cartilage degeneration and delay HA degeneration in hemophilic arthritis.

2. The use according to claim 1, characterized in that In the presence of blood, dihydroartemisinin DHA delays chondrocyte degeneration by inhibiting the level of ROS in chondrocytes; in verification, DHA delays the degeneration of HA by delaying the level of ROS in chondrocytes.

3. The use according to claim 1, characterized in that The active ingredient of the chondrocyte antioxidant drug is dihydroartemisinin.

4. The use according to claim 1, characterized in that: The chemical formula of dihydroartemisinin DHA is C 15 H 24 O5, the structural formula is as follows:

5. The use according to claim 1, characterized in that The drug is administered orally.

6. The use according to claim 1, characterized in that: The drug is a drug that reduces the level of reactive oxygen species (ROS) in chondrocytes of F8 factor-deficient model mice and delays chondrocyte degeneration.

7. The use according to claim 1, characterized in that: The effective dose of dihydroartemisinin in delaying chondrocyte degeneration is 10 mg / kg.

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